Nuclear Factor kappa Bs (NF-KBs) are activated by over 200 stimuli by different mechanisms of signal transduction. These molecular signaling mechanisms are only partially understood for a limited number of the stimuli. Activation of NF-xBs in response to many stimuli is regulated by IKB kinase (IKK). A multi component complex consisting of alpha, beta, and gamma subunits. IKKa and IKKb are the catalytic kinase subunits, which form homo-and heterodimers. IKKy, which associates to IKKa and IKKbeta is a regulatory component required for signal-induced IKK activation. The molecular signaling mechanisms that regulate IKK activation in response to different stimuli remain unknown. In this application, we propose that interaction of IKKy with the C-termini of IKKalpha and IKKbeta is regulated by phosphorylation in a signal-dependent manner. We further propose that regulation of this interaction plays a role in activation of IKK by different stimuli. We have identified serine residues in the gamma-binding domain (yBD) of IKKbeta that are phosphorylated by a novel kinase. Phosphorylation of the serines in the yBD reduces the interaction of IKKbeta with IKKy. We suggest that modulating the affinity of the interaction between the catalytic subunits and IKKy maybe an important regulatory step for regulation of IKK. Based on data presented in this application, we propose a model for differential activation of IKK (Figs 2 and 11). In this application, we will test the model with the following Specific Aims: Aim 1: Test the hypothesis that phosphorylation of the carboxy termini (the IKKy interaction domains) of lKKalpha and IKKbeta regulates the IKK's activity through modulation of IKKy interaction with the catalytic subunits in a stimulus-dependent manner. Aim 2: Test the hypothesis that IKKgamma interacts with more than one region of IKKalpha and IKKbeta, and determine the interaction domains. Aim 3: Biochemically purify and molecularly clone yBD kinase, and characterize its regulation by different IKK activators, and test whether yBD kinase is sufficient to maintain IKK in its lowest, basal level state of activity.